Cytotoxicity Effects And Metabolic Mechanisms Of Low Dose Tetrabromobisphenol A(TBBPS) Exposure

Brominated flame retardants(BFR),as additives for stop fire,are widely used in the production of commercial products such as plastics,textiles,electronic and electrical equipment,light socket,water pipe,and thermal insulation cotton.Tetrabrominated bisphenol A(TBBPA)is one of the most common typical flame retardants,accounting for about 60% of the market.However,in the process of production and use,part of TBBPA could be released into the environment.TBBPA has been detected in air samples from the electronics industry,river sediments and sewage sludge samples.Some studies have reported that TBBPA can accumulate in the serum,umbilical cord blood and breast milk of pregnant and lactating women.And since the half-life of TBBPA has been reported to be about two days in humans,bioaccumulation suggests that mothers are exposed to TBBPA for a long period of time in daily life.At the same time,the concentrations of TBBPA detected in the serum of infants was 2 to 5times higher than that detected in mothers,suggesting a particular risk of TBBPA exposure during human development.Current studies have shown that TBBPA affects neurodevelopment,disrupts endocrine system,and increases the risk of tumor development.In this study,human hepatocellular carcinoma cell line Hep G2 was used to carry out the cell exposure experiment by using the residual TBBPA concentration in human plasma,and the possible health risks caused by long-term exposure to TBBPA in occupational population were analyzed.Analyses were carried out from two aspects of cell physiological toxic effects and changes in cell transcriptome expression.The possible health risks caused by long-term exposure to TBBPA in occupational population were analyzed by cell physiological activity index measurement,cell damage measurement,cell oxidative stress level measurement,apoptotic cell proportion assessment and eukaryotic transcriptome sequencing.We mainly studied the cellular physiological effects of long-term exposure to low concentrations of TBBPA at the level of people with occupational exposure,analyzed the interference of TBBPA on transcriptome regulation process through sequencing results,and carried out pathway verification and mechanism analysis.The main research contents and results of this paper are as follows:Firstly,the physiological toxic effects of cells were analyzed from the perspectives of cell proliferation,injury,oxidative stress and apoptosis,and the results showed that: Long-term exposure to low concentration of TBBPA significantly promoted the proliferation and migration activity of Hep G2 cells.The proliferation activity of cells exposured to 1 n M TBBPA for 5 generations was 1.56 times that of control group.In addition,long-term exposure to TBBPA can double the level of reactive oxygen species(ROS)released by mitochondria and promote the up-regulation of adenosine monophosphate-activated protein kinase(AMPK)gene expression,thereby promoting the ability of cell proliferation and survival.ROS can also mediate the process of cell apoptosis through the mitochondrial membrane potential(MMP),which enhances the mitochondrial activity,causing cell in hyperpolarization state,inhibits cell early apoptosis process.However,at the same time,the expression of cysteine-containing aspartic acid proteolytic enzymes caspase-3 and caspase-9,which are responsible for regulating the release of apoptosis-related chemical signals was upregulated,ultimately leading to an increase in the overall proportion of apoptotic cells.These results indicated that long-term exposure to occupational exposure dose of TBBPA not only activated the survival self-help mechanism of Hep G2 cells,promote the cell proliferation activity and mitochondrial hyperpolarization,but also caused toxic damage to Hep G2 cells,cellular ROS production is on the high side,induced the release of chemical signals apoptosis protein gene expression level,leading to apoptosis cells than to rise.Secondly,RNA sequencing and result analysis of Hep G2 cells exposed to long-term TBBPA were carried out.The analysis results showed that: Long-term exposure to low-dose TBBPA could interfere with the regulation of cell gene expression.The expression and regulation of genes related to cell physiological processes and cell components are affected.It also interferes with the expression of genes related to lipid metabolism,vitamin metabolism,nucleotide metabolism and protein metabolism of cells.Through q-PCR detection,it is confirmed that the RAS signaling pathway is activated by growth factors,mediating the cell detoxification mechanism,and then prolonging the life of cancer cells.At the same time,the eukaryotic transcriptome sequencing results show that the endocrine disrupting effect of TBBPA is related to its induction of upregulation of thyroid hormone and parathyroid hormone receptors,q-PCR assay proved that after exposed to TBBPA for five generations,expression levels of thyroid hormone and parathyroid hormone pathway related genes in cells were upregulated compared with the cells exposed to TBBPA for 1 generation,and the upregulation level is proportional to the exposure period.After exposed to TBBPA and TR Antagonist 1 for 5 generations,the expression of thyroid hormone and parathyroid hormone receptors was significantly reduced,and the activation of Ras pathway was also inhibited.The expression level of RAS gene was down-regulated,which proved that the toxic effect of low concentration TBBPA was directly related to the thyroid hormone receptors.In summary,a series of toxic and damaging effects of typical brominated flame retardants on human metabolic system were studied in this paper from two aspects of cell physiological toxic effects and gene expression level.The study found that in the case of long-term exposure to low dose of TBBPA,human hepatocellular carcinoma cells Hep G2 will produce oxidative stress,causing toxic damage,but also activate its own survival mechanism,induce the high expression of oncogenes,and induce the high expression of thyroid hormone and parathyroid hormone receptor-related genes generation by generation.The research results obtained in this paper reveal the health exposure risk of TBBPA at the daily exposure level of human body,and provide certain reference significance for the study of long-term exposure toxicity and toxic action mechanism of typical toxic pollutants to human body.